Voltage-gated ion channels confer electrical excitability on neurons and muscle cells and thereby play essential roles in the physiology of brain, heart, and skeletal muscle. Disruption of channel function or expression results in neurological, muscular, and cardiovascular diseases. The goal of this project is to determine the molecular mechanism by which voltage controls channel activity. In the proposed research, we will use the Shaker K+ channel to investigate fundamental aspects of voltage-dependent activation that are poorly understood or previously unrecognized. The Specific Aims of this proposal are: 1) To probe the structural environment of voltage-sensing residues in S4 during activation. We will test the hypotheses that S4 arginine residues interact with conserved acidic residues in S2 and S3 in closed conformations and follow a common pathway as they traverse the transmembrane electric field during activation. 2) To investigate S3b motion during voltage-dependent activation. A major controversy exists between different models for the mechanism of activation. Does S4 move independently or in conjunction with S3b? We will test the range of S3b motion relative to its environment by determining its proximity to S2, a stationary component of the voltage sensor domain, during activation. 3) To test the hypotheses that F290 serves as the physical barrier between internal and external gating crevices and is energetically coupled to charge-moving S4 arginine residues during activation. In the original version of this application, we proposed to test the hypothesis that F290 facilitates passage of charge-moving S4 residues across the electric field. New results from the laboratory of Rod MacKinnon strongly support this proposal. In the revised Aim we will investigate 3 key questions about the role of F290 during activation. First, what are the contributions of F290, I241 in S1, and I287 in S2 to the lid that excludes water from the barrier where the electric field is focused? Second, does R1 cross F290 to reside below the barrier in the resting state? This idea, which is consistent with MacKinnon's results, is contrary to previous experimental and computational analyses of R1's position at rest. Third, is F290 energetically coupled to each charge-moving residue in S4? This question has important implications for the mechanism of charge transfer across the field and the number and structure of intermediate closed conformations in the activation pathway. To accomplish these Specific Aims, we will determine the effects of experimental perturbations on ionic and gating currents and the fluorescent intensity of reporter fluorophores that detect different phases of the activation mechanism. In collaboration with Dr. Benoit Roux, experimental constraints identified in the research will be used to generate low resolution structural models of the resting and, if possible, intermediate closed conformations. MD simulations will be used to assess changes in local protein dynamics that might underlie experimental results. In this work, we will build on our notable success in identifying specific, short range structural interactions, including state-dependent interactions, in voltage-gated K+ channels. PUBLIC HEALTH RELEVANCE: Voltage-gated ion channels confer electrical excitability on neurons and muscle cells and thereby play essential roles in the physiology of brain, heart, and skeletal muscle; disruption of channel function or expression results in neurological, muscular, and cardiovascular diseases. The goal of this research is to determine the mechanism by which voltage controls channel activity. We will study the function of the barrier that voltage-sensing residues cross during activation. An increase in the permeability of this barrier is a newly- discovered cause of human genetic diseases; the proposed research may lead to new therapeutic approaches to seal the barrier in these diseases.